Reagents and methods for the detection and quantification of thyroxine in fluid samples

ABSTRACT

Immunoassay methods and reagents for the specific quantification of thyroxine in a test sample are disclosed employing antibodies prepared with thyroxine derivatives of the formula: ##STR1## wherein P is an immunogenic carrier material and X is a linking moiety. The present invention also describes the synthesis of unique labelled reagent of the formula: ##STR2## wherein Q is a detectable moiety and W is a linking moiety, preferably fluorescein or a fluorescein derivative.

This application is a division of U.S. patent application Ser. No.08/275,064, filed Jul. 13, 1994, now U.S. Pat. No. 5,593,896, which is acontinuation-in-part application of U.S. patent application Ser. No.859,775 of P. G. Mattingly, filed Mar. 30, 1992, now U.S. Pat. No.5,352,803, entitled "5(6)-Methyl Substituted Fluorescein Derivatives".

FIELD OF THE INVENTION

The present invention relates to the immunoassay quantification ofthyroxine in a test sample. In particular, the present invention relatesto immunogens, antibodies prepared from such immunogens, and labelledreagents for the specific quantification of thyroxine in a test sample,preferably for use in fluorescence polarization immunoassays.

BACKGROUND OF THE INVENTION

The amino acid 3,5,3',5'-tetraiodo-L-thyronine, commonly calledtbyroxine and often referred to as T₄, is the predominant iodothyroninesecreted from the thyroid gland. T₄ is responsible for regulatingdiverse biochemical processes throughout the body, which are essentialfor normal metabolic and neural activities. The measurement of serum T₄concentration has become the common initial test in the diagnosis ofaltered thyroid function. Several conditions other than. thyroid,disease may cause abnormal serum levels of T₄. Among these arepregnancy, estrogenic or androgenic steroids, oral contraceptives,hydantoins and salicylates, stress, hyper- and hypoproteinemia, andconditions (hereditary or acquired) which cause alterations in serumlevels of thyroid binding globulin (TBG), the major serum T₄ transportsystem.

The concentration of thyroxine in the bloodstream is extremely low andcan only be detected with very sensitive techniques. Approximately 0.05%of the total circulating thyroxine is physiologically active (i.e. freethyroxine). The remaining circulating thyroxine is bound to proteins,primarily thyroxine binding globulin (TBG). Thyroxine will also bind toother binding proteins, particularly, thyroxine binding pre-albumin andalbumin. Early T₄ determinations were indirect measurements of theconcentration of protein-bound or butanol-extractable iodine in serum.Later, competitive protein binding (CPB) assays were developed. Morerecently, radioimmunoassay procedures have been developed which use bothpolyclonal and monoclonal antibodies, such as disclosed in U.S. Pat.Nos. 4,636,478 and 4,888,296 to Siebert et al., which discloseradioimmunoassay for thyroxine using specific monoclonai antibodiesrecognizing L-thyroxine. In general, radioimmunoassay procedures in theart measure counts of radioactivity which are related to the binding ofthe antibody to L-thyroxine.

D-thyroxine is a non-naturally occurring isomer of thyroxine. Both L-and D-thyroxine are represented by Formula 1 below: ##STR3##

More recently, fluorescent polarization techniques have been used toassay for thyroxine. Fluorescent polarization techniques are based onthe principle that a fluorescent labelled compound when excited bylinearly polarized light will emit fluorescence having a degree ofpolarization inversely related to its rate of rotation. Therefore, whena fluorescent labelled tracer-antibody complex is excited with linearlypolarized light, the emitted light remains highly polarized because thefluorophore is constrained from rotating between the time light isabsorbed and emitted. When a "free" tracer compound (i.e., unbound to anantibody) is excited by linearly polarized light, its rotation is muchfaster than the corresponding tracer-antibody conjugate and themolecules are more randomly oriented, therefore, the emitted light isdepolarized. Thus, fluorescent polarization provides a quantitativemeans for measuring the amount of tracer-antibody conjugate produced ina competitive binding immunoassay.

U.S. Pat. Nos. 4,510, 251 and 4,614,823, to Kirkemo et al., disclosefluorescent polarization assays for ligands using aminomethylfluoresceinderivatives as tracers, and the aminomethylfluorescein derivatives,respectively. U.S. Pat. No. 4,476,229, to Fino et al., disclosessubstituted carboxyfluoresceins, including those containing a thyroxineanalog, for use in fluorescence polarization immunoassays. U.S. Pat. No.4,668,640, to Wang et al., discloses fluorescence polarizationimmunoassay utilizing substituted carboxyfluoresceins. Example IX ofWang et al. discloses a method for making aL-thyroxinecarboxyfluorescein conjugate of the following formula:##STR4##

Both the Wang et al. and Fino et al. patents present a conjugate inwhich a carboxyfluorescein is directly attached to the amino group ofthe thyroxine via an amide bond.

Examples of commercially available fluorescence polarizationimmunoassays (FPIA) for thyroxine are: IMx®, TDx®, and TDxFLx™ T₄ assays(Abbott Laboratories, Abbott Park, Ill. Hereinafter also referred to asthe "commercially available Abbott T₄ assay(s)" or "commerciallyavailable T₄ assay(s)") which include reagent systems for thequantitative measurement of total (i.e. free and protein bound)thyroxine present in a serum or plasma sample. All of these assays usethe same fluorescent T₄ derivative as a tracer, which is labelled with acarboxyfluorescein (hereinafter also referred to as the "commerciallyavailable T₄ tracer" or "commercially available tracer"); the same sheeppolyclonal antibodies against thyroxine (hereinafter also referred to as"commercially available T₄ antibodies", or "commercially availableantibodies"); and the same reagent for removing the protein from theprotein bound thyroxine in order to release the thyroxine for assay.

FPIAs have an advantage over radioimmunoassay (RIA) in that there are noradioactive substances to dispose of and FPIAs are homogenous assaysthat can be easily performed. However, it has been reported that thecommercially available Abbott TDx® T₄ assay showed a low level of T₄which did not conform to radioimmunoassay measurement and the clinicalsymptoms of hypothyroidism. See Levine, S. et al., Clin. Chem., 36 (10):1838-1840 (1990).

SUMMARY OF THE INVENTION

The present invention provides unique antibody reagents and labelledreagents for the quantification of thyroxine in a test sample. Thepresent invention also provides synthetic procedures for preparing thelabelled reagents, and for preparing immunogens which are employed forthe production of the antibody reagents. According to the presentinvention, the labelled reagents and the antibody reagents offer anadvance in the art beyond previously known procedures when used in animmunoassay for the quantification of thyroxine in a test sample.According to a preferred embodiment of the present invention, thelabelled reagent and the antibody reagent are used in a fluorescencepolarization immunoassay which combines specificity with the speed andconvenience of homogeneous methods to provide a reliable quantificationof thyroxine in a test sample and to avoid interference from endogenousimmunoglobulin G (hereinafter referred to as "lgG") produced by somehumans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the synthetic pathway for making an immunogen of thepresent invention by coupling L-thyroxine to bovine serum albumin (BSA)according to the synthetic method of the present invention.

FIG. 2 illustrates the synthetic pathway for the preparation of afluorescent tracer of the present invention according to the syntheticmethod of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, the specific quantification ofthyroxine is accomplished by first contacting a test sample with alabelled reagent or tracer and an antibody reagent, eithersimultaneously or sequentially in either order, and then measuring theamount of the labelled-reagent which either has or has not participatedin a binding reaction with the antibody reagent as a function of theamount of thyroxine in the test sample.

The test sample can be any naturally occurring body fluid or tissue, oran extract or dilution thereof, and includes, but is not intended to belimited to whole blood, serum, plasma, urine, saliva, cerebrospinalfluid, brain tissue, feces, and the like.

In particular, the present invention relates to immunogens, antibodiesprepared from such immunogens, and labelled reagents for use influorescence polarization immunoassays (FPIA) for the specificquantification of thyroxine.

Throughout this application, the chemical structures shown in theformulae can be either the L or D isomer or a combination of L and Disomers. However, in all the formulae, L isomers am the most preferred.

Antibodies, both polyclonal and monoclonal antibodies, of the presentinvention are produced with immunogens of the following general formula:##STR5## wherein P is an immunogenic carrier material and X is a linkingmoiety. The terms linking moeity, tether, spacer, spacer arm, and linkerare used interchangeably and are meant to define any covalently boundchemical entity that separates one defined substance (such as a hapten)from a second defined substance (such as an immunogenic carrier ordetectable moiety).

In the present invention, X is a linking moiety preferably consisting offrom 0 to 50 carbon and heteroatoms, including not more than tenheteroatoms, arranged in a straight or branched chain or cyclic moietyor any combination thereof, saturated or unsaturated, with the provisosthat: (1) not more than two heteroatoms may be directly linked, (2) Xcannot contain --O--O-linkages, (3) the cyclic moieties contain 6 orfewer members, and (4) branching may occur only on carbon atoms.Heteroatoms may include nitrogen, oxygen, sulfur and phosphorous.Examples of X are: alkylene, aralkylene and alkylene substitutedcycloalkylene groups. It shall be noted that, according to thedefinition herein, X can be zero, i.e. when the carbon and heteroatomare zero. If X=0, then no linking moiety exists, which indicates that Pis directly linked to the thyroxine derivative in Formula 2.

As would be understood by one skilled in the art, the immunogeniccarrier material P, can be selected from any of those conventionallyknown in the art, and in most instances will be a protein orpolypeptide, although other materials such as carbohydrates,polysaccharides, lipopolysaccharides, poly(amino) acids, nucleic acids,and the like, of sufficient size and immunogenicity can also beemployed. Preferably, the immunogenic carrier material is a protein suchas bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH),thyroglobulin, and the like.

In the preferred immunogen, P is bovine serum albumin (BSA) and X is--NH(CH₂)₅ C(═O)--. The preferred immunogen is shown below: ##STR6##

The most preferred thyroxine immunogen is the L isomer of Formula 3.TheFormulae 2 and 3 are not limited to one to one conjugates of thyroxineand the immunogenic carrier, as one skilled in the art would realize.The ratio of thyroxine derivative to immunogenic carrier is defined bythe number of chemically available functional groups on the immunogeniccarrier and controlled by the ratio of the two materials in thesynthesis. The degree of substitution on P by the thyroxine derivativecan vary between 1 to 100% of the available functional groups on theimmunogenic carrier. The level of substitution is preferably between 10%to 95%; and more preferably, between 15% to 85%.

Labelled reagents of the present invention have the following generalformula: ##STR7## wherein Q is a detectable moiety, preferably afluorescent moiety; and W is a linking moiety. In the preferred labelledreagent, Q is a fluorescein derivative chosen from the group consistingof 4'-aminomethylfluorescein, 5-aminomethylfluorescein,6-aminomethyifluorescein, 5-carboxyfiuorescein, 6-carboxyfiuorescein, 5and 6-aminofluorescein, thioureafiuorescein, andmethoxytriazinolyl-aminofluorescein. W is a linking moiety preferablyconsisting of from 0 to 50 carbon and heteroatoms, including not morethan ten heteroatoms, arranged in a straight or branched chain or cyclicmoiety or any combination thereof, saturated or unsaturated, with theprovisos that: (1) not more than two heteroatoms may be directly linked,(2) W cannot contain --O--O-linkages, (3) the cyclic moleties contain 6or fewer members, and (4) branching may occur only on carbon atoms.Heteroatoms may include nitrogen, oxygen, sulfur and phosphorous. Thespecific chemical structure of W can be the same or different from thatof the X of Formula 2. Examples of W are: alkylene, aralkylene andalkylene substituted cycloalkylene groups. It shall be noted that,according to the definition herein, W can be zero, i.e. when the carbonand heteroatom are zero. If W=0, then no linking moiety exists, whichindicates that Q is directly linked to the thyroxine derivative inFormula 4.

The preferred labelled reagent is of the following formula: ##STR8##

The most preferred labelled reagent is the L isomer of Formula 5. Anexample of a process for preparing the 5 methyl-substituted fluoresceinderivative of Formula 5 is disclosed in U.S. patent application Ser. No.859,775, of P. G. Mattingly, filed Mar. 30, 1992, entitled "5(6)-MethylSubstituted Fluorescein Derivatives" which is incorporated herein byreference.

The present invention has a surprising feature. It is known to one ofordinary skill in the art that when preparing specific antibodies andcomplementary labelled haptens (as the labelled reagents), one needs toconsider the chemical structure of both the immunogen used to elicit theantibody response and the labelled hapten. Traditionally, one attachesthe hapten to the carrier protein through a site on the hapten that isremote from the unique features of the hapten that are critical forachieving selective antibodies. Likewise, when preparing a labelledhapten able to bind to such antibodies, it is customary to attach thelabel to the hapten through the same site as the carrier protein. Onereason behind such an approach is that the carrier protein maysterically block access of the immune system to that part of the hapten.Normally, the complementary labelled hapten is synthesized by attachingits label to the same site on the hapten as the immunogen uses forattachment of its carrier protein, so as not to interfere with antibodybinding to the critical features of the hapten.

Therefore, it is surprising and unexpected to find that the thyroxineimmunogen and labelled thyroxine of the present invention, which werederived from different sites of attachment on thyroxine, lead todevelopment of specific antibodies to thyroine and an excellent assaywith improved quantification of thyroxine.

Specifically, in the present invention, the immunogen was prepared froma thyroxine molecule which was conjugated to the carrier protein via thecarboxylic acid terminal of thyroxine; whereas the labelled thyroxinereagent was prepared by attachment of the label at the amino terminal ofthyroxine.

Additionally, as mentioned in the "Background of the Invention", theWang et al. and Fino et al. patents present a conjugate in which acarboxyfluorescein is directly attached to the amino group of thethyroxine via an amide bond. In the current invention, the detectablemoiety is attached, via a linking moiety to N-carboxymethyl-L-thyroxine,wherein the original amino group of the thyroxine is a secondary amine,not an amide. Further, unlike Wang et al and Fino et al., the syntheticmethod of the current invention requires a multistep treatment ofthyroxine which makes extensive use of orthogonal protecting groups toarrive at the desired structure.

Preparation of the Immunogen

The general structure of the immunogen is as shown in Formula 2, where Xis a linking moiety and P is an immunogenic carrier. Immunogens ofFormula 2 may be produced according to the following scheme: ##STR9##

The N-acetyl-L-thyroxine (I) is coupled, according to methods known tothose skilled in the art, with a bifunctional linker designated v-X-ywherein v- and -y are functional groups, one of which can react with thecarboxylate of N-acetyl-L-thyroxine (I) and the other with chemicallyavailable functional groups on P. X is the linking moiety. Manybifunctional linkers are known to one skilled in this art. For example,heterobifunctional linkers are described in U.S. Pat. No. 5,002,883 toBieniarz, et al, hereby incorporated by reference. Theseheterobifunctional linkers are preferred in some cases due to thespecificity of their ends for one functional group or another. Likewise,for convenience in the synthesis, protected forms of the functionalgroups v- and -y, well known to those skilled in the art (see e.g. T. W.Greene and P. G. M. Wutts, Protective Groups in Organic Synthesis, 2nded. 1991, John Wiley and Sons, hereby incorporated by reference) may beused and deprotected at the desired time.

Generally, in the preparation of immunogens of the present invention, vis selected from the group consisting of --OH, -halogen (e.g. --Cl,--Br, --I), --SH, and --NHR'--. R' is selected from H; alkyl, aryl,substituted alkyl and substituted aryl; y is chosen from the groupconsisting of: hydroxy (--OH), carboxy (--C(═O)OH), amino (--NH₂),aldehyde (--CH(═O)), and azido (--N₃). X is a linking moiety preferablyconsisting of from 0 to 50 carbon and heteroatoms, including not morethan ten heteroatoms, arranged in a straight or branched chain or cyclicmoiety or any combination thereof, saturated or unsaturated, with theprovisos that: (1) not more than two heteroatoms may be directly linked,(2) X cannot contain --O---linkages, (3) the cyclic moieties contain 6or fewer members, and (4) branching may occur only on carbon atoms.Heteroatoms may include nitrogen, oxygen, sulfur and phosphorous.Examples of X are: alkylene, aralkylene and alkylene substitutedcycloalkylene groups. It shall be noted that, according to thedefinition herein, X can be zero, i.e. the carbon and heteroatom arezero. If X=0, then no linking moiety exists, which indicates that P isdirectly linked to the thyroxine derivative in Formula 2.

Reaction of the N-acetyI-L-thyroxine (5) with v-X-y produces tetheredintermediate compound (II) having linking moiety X with a functionalgroup y. The functional group -y, can be reacted in any of several ways,known to those skilled in the art, with the functional groups on animmunogenic carrier. It is frequently preferable to form amide bonds,which typically are quite stable. Amide bonds are formed by firstactivating the carboxylic acid moiety y═(--C(═O)OH)! of the spacer armby reaction with an activating reagent such as1,3-dicyclohexylcarbodiimide and an additive such asN-hydroxysuccinimide. The activated form is then reacted with a bufferedsolution containing the immunogenic carrier materials. Alternatively,the carboxylic acid group may be converted, with or without isolation,into a highly reactive mixed anthydride, acyl halide, acyl imidazolide,or mixed carbonate and then combined with the immunogenic carriermaterials. One of ordinary skill in the art will recognize that thereare many reagents that can be used to form amide bonds other than thoselisted.

A spacer arm with a terminal amine (y═--NH₂) functionality can betransformed into a highly reactive N-hydroxysuccinimide urethane byreaction with N,N'-disuccinimidyl carbonate in a suitable solvent, suchas acetonitrile or dimethylformamide. The resultant urethane is thenreacted with the immunogenic carrier materials in a buffered, aqueoussolution to provide an immunogen.

A spacer arm with a terminal aldehyde functionality y═--CH(═O)! can becoupled to the immunogenic carrier materials in a buffered, aqueoussolution and in the presence of sodium cyanoborohydride, by reductiveamination according to methods known to those skilled in the art.

Alternatively, spacer arms containing an alcohol group y═--OH! can becoupled to the immunogenic carrier materials by first reacting it withphosgene or phosgene equivalent, such as di or triphosgene orcarbonyldiimidazole, resulting in the formation of a highly reactivechloroformate or imidazoloformate derivative (usually withoutisolation). The resultant active formate ester is then reacted with theimmunogenic carrier materials in a buffered, aqueous solution to providean immunogen.

Alternatively, when y═--N₃, the tethered intermediate can be coupled toP by photolysis in aqueous buffered solution.

The preferred immunogen of Formula 3 is thus prepared according to thescheme of FIG. 1. L-thyroxine (1) sodium salt is converted toN-acetyl-L-thyroxine (5); the carboxyl group of N-acetyl-L-thyroxine (5)is activated with dicylcohexylcarbodiimide and N-hydroxysuccinimide (thebold-faced numerals contained in parenthesis refer to the structuralformulae used in FIG. 1). Further reaction with the linker,6-aminocaproic acid v═--NH₂, X═--(CH₂)₅ -, y═--CO₂ H! gives the tetheredintermediate 6, X═--(CH₂)₅ -, y═--CO₂ H!. The y-group is then activatedwith dicylcohexylcarbodiimide and N-hydroxysuccinimide and coupled to P.Those skilled in the art will recognize that other methods for peptidebond formation could be employed with equal success.

In a manner analogous to immunogens, spacer arms can be conjugated tosolid supports having functional groups such as amino, hydroxyl orcarboxyl groups that are reactive in a complementary sense with reactivegroups on the spacer arm. The result is a solid phase which can be usedto separate or purify antibodies against the hapten.

Thus the above thyroxine derivatives can be coupled to immunogeniccarrier materials P by various conventional techniques known in the art.

Production of Antibodies

The immunogens according to the present invention are used to prepareantibodies, both polyclonal and monoclonal, according to methods knownin the art, for use in an immunoassay system according to the presentinvention. Generally, a host animal, such as a rabbit, goat, mouse,guinea pig, or horse is injected at one or more of a variety of siteswith the immunogen, normally in a mixture with an adjuvant. Furtherinjections are made at the same site or different sites at regular orirregular intervals thereafter with bleedings being taken to assessantibody titer until it is determined that optimal titer has beenreached. The antibodies are obtained by either bleeding the host animalto yield a volume of antiserum, or by somatic cell hybridizationtechniques or other techniques known in the art to obtain monoclonalantibodies, and can be stored, for example, at -20° C. Besides wholeimmunoglobulins, antibodies herein include antigen binding fragments ofthe immunoglobulins. Examples of these fragments are Fab, F(ab')₂ andFv. Such fragments can be produced by known methods.

It is to be noted from Example 4 that the replacement of thecommercially available T₄ tracer with the labelled reagent of thepresent invention alone improves the performance of a thyroxine assay.Thus, the assays or kits can use the labelled reagents of the presentinvention with antibodies, whether polyclonal or monoclonal, whichrecognize both thyroxine and the labelled reagents, and which arepreferably antibodies that are raised by the immunogens of Formulae 2and 3. Additionally, to enable the performance of competitiveimmunoassays such as FPIA, the tracers and thyroxine must be able tocompetitively bind to the antibodies. Since the test samples wouldmostly be biological samples, though the antibodies may bind bothisomers of thyroxine, it is preferable that the antibodies preferablybind L-thyroxine. Similarly, the immunogens are preferably derivativesor analogs of L-thyroxine. The labelled reagents preferably do not bindor significantly bind endogenous immunoglobulins which may be found inthe test sample, i.e. antibodies that are not intended to bind thelabelled reagents, such that the binding interferes with the accuracy ofthe assay. In Example 4 below, these immunoglobulins are immunoglobulinsG (IgG).

Preparation of the Labelled Reagent

The following describes the method for synthesizing the labelledreagents of the present invention. These labelled reagents can besynthesized from thyroxine by: (a) differentially protecting thecarboxylic acid, α-amino and phenolic groups of thyroxine (according tothe method as shown in, e.g. T. W. Greene and P. G. M. Wutts, ProtectiveGroups in Organic Synthesis, 2nd ed. 1991, John Wiley and Sons, herebyincorporated by reference); then (b) selectively deprotecting theα-amino group of the thyroxine derivative; next (c) selectivelycarbalkoxymethylating the α-amino group; followed by (d) selectivelydeprotecting the α-N-carboxymethyl group and the phenolic group of thethyroxine derivative; then (e) activating the α-N-carboxymethyl group;next (f) coupling the activated α-N-carboxymethyl group of the thyroxinederivative with a bifunctional linking moiety, then (g) coupling with adetectable moiety; and finally (h) deprotecting the carboxylic acidgroup of the labelled reagent. One skilled in the art would alsorecognize that steps f and g could be combined; the detactable moietycould be coupled to the bifunctional linking moiety before coupling tothe thyroxine derivative of step e.

More specifically, the labelled reagent can be synthesized by: (a) (i)reacting a sodium salt of thyroxine with 9-fluorenylmethoxycarbonylchloride (FMOC-Cl) to protect the amino group followed by (ii)protecting the phenolic functionality of the resulting thyroxinederivative by acetylation, then (iii) protecting the carboxylic group ofthe N-FMOC-O-acetyl-thyroxine as the tert-butyl ester; followed by (b)removing the FMOC protective group to give t-butyl O-acetyl-thyroxine;next (c) alkylating areinc group of t-butyl O-acetyl-thyroxine withbromoacetic acid ethyl ester to give t-butylO-acetyl-N-carboethoxymethyl-thyroxine; then (d) hydrolysing the ethylester and acetyl groups of t-butylO-acetyl-N-carboethoxymethyl-thyroxine with sodium hydroxide in methanoland obtaining t-butyl N-carboxymethyl-thyroxine in the same process; and(e) activating the t-butyl N-carboxymethyl-thyroxine withdicylcohexylcarbodiimide and N-hydroxysuccinimide; then (f) reacting thethyroxine derivative with 5-aminomethylfluorescein to give t-butylN-(5-carboxamidomethylfluoresceinyl-methyl)-thyroxine; followed by (g)hydrolysing the t-butyl ester with trifluoroacetic acid to give thelabelled reagent. This method is exemplified in the synthesis of thepreferred labelled reagent as shown in FIG. 2.

Preferably, the above synthetic methods am used to produce the labelledreagents of Formulae 4 and 5, and more preferably the L structures ofthese formulae.

Thyroxine Assay Utilizing Fluorescence Polarization Immunoassay

The concentration or level of thyroxine in a test sample can beaccurately quantified in a fluorescence polarization immunoassay (FPIA)by employing the reagents of the present invention. To perform a FPIAfor the specific quantification of thyroxine, calibration curves usingknown amount, of thyroxine were generated for measuring the thyroxine ina sample.

According to the present invention, it has been unexpectedly andsurprisingly found that superior fluorescence polarization immunoassayassay results for the quantification of thyroxine are obtained whenemploying the L isomer of the fluorescent labelled reagent (or tracer)of Formula 5.

In particular, it was unexpectedly and surprisingly found that the useof this labelled reagent was critical for the avoidance of discrepantresults. This represents an advance over the commercially availableAbbott T₄ assays for the specific quantification of thyroxine. Moregenerally, the tracer can be of Formula 4. When performing afluorescence polarization immunoassay for the specific quantification ofthyroxine as described herein, the detectable moiety component of thetracer is a fluorescent moiety such as fluorescein, aminofluorescein,carboxyfluorescein, and the like, preferably 5 and6-aminomethylfluorescein, 5 and 6-aminofluorescein,6-carboxyfluorescein, 5-carboxyfluorescein, thioureafluorescein, andmethoxytriazinolyl-aminofluorescein, and similar fluorescentderivatives. The fluorescent tracer can be used in combination with anantibody which is capable of binding both the tracer and T₄. For acompetitive immunoassay the tracer and T₄ must be able to competitivelybind to the antibody. For the quantification of thyroxine, the antibodyreagent comprises antibodies which are capable of binding to orrecognizing thyroxine wherein the antibodies are preferably producedwith an immunogen of Formula 2, and more preferably that of Formula 3.

The amount of tracer bound to the antibody varies inversely to theamount of thyroxine present in the test sample. Accordingly, therelative binding affinities of thyroxine and the tracer to the antibodybinding site are important parameters of the assay system.

Generally, fluorescent polarization techniques are based on theprinciple that a fluorescent tracer, when excited by plane polarizedlight of a characteristic wavelength, will emit light at anothercharacteristic wavelength (i.e., fluorescence) that retains a degree ofthe polarization relative to the incident stimulating light that isinversely related to the rate of rotation of the tracer in a givenmedium. As a consequence of this property, a tracer substance withconstrained rotation, such as in a viscous solution phase or when boundto another solution component such as an antibody with a relativelylower rate of rotation, will retain a relatively greater degree ofpolarization of emitted light than if in free solution.

When performing a fluorescent polarization immunoassay for the specificquantification of thyroxine according to the present invention, a testsample suspected of containing thyroxine is contacted with antiserum ormonoclonal antibodies prepared with immunogens according to the presentinvention, in the presence of labelled reagent of the present invention,which is capable of producing a detectable fluorescence polarizationresponse to the presence of antiserum or monoclonal antibodies preparedwith immunogens according to the present invention. Plane polarizedlight is then passed through the solution to obtain a fluorescentpolarization response and the response is detected as a measure ofamount of thyroxine present in the test sample.

The thyroxine derivatives of the present invention are employed toprepare immunogens by coupling them to conventional carrier materials,and subsequently used to obtain antibodies. The thyroxine derivatives ofthe present invention are also used to prepare labelled reagents whichserve as the detection reagents in immunoassays for quantifyingthyroxine in a test sample.

The fluorescence polarization assays can be conducted in commerciallyavailable automated instruments such as: IMx®, TDx®, and TDxFLx™ (AbbottLaboratories).

Other Assay Formats

In addition to fluorescence polarization immunoassays, various otherimmunoassay formats can be followed for the quantification of thyroxineaccording to the present invention. Such immunoassay system formatsinclude, but are not limited to competitive and sandwich assaytechniques. Generally, such immunoassay systems depend upon the abilityof an immunoglobulin, i.e., a whole antibody or fragment thereof, tobind to a specific analyte from a test sample wherein a labelled reagentcomprising an antibody of the present invention, or fragment thereof,attached to a label or detectable moiety is employed to determine theextent of binding. Such labels or detectable moieties include, but arenot intended to be limited to, enzymes, radiolabels, biotin, toxins,drugs, haptens, DNA, RNA, liposomes, chromophores, chemiluminescens,colored particles and colored microparticles, fluorescent compounds suchas aminomethylfluorescein, 5-carboxyfluorescein, 6-carboxyfluorescein,aminofluorescein, thioureafluorescein, andmethoxytriazinolyl-aminofluorescein, and the like fluorescentderivatives.

Typically, the extent of binding in such immunoassay system formats isdetermined by the amount of the detectable moiety present in thelabelled reagent which either has or has not participated in a bindingreaction with the analyte, wherein the amount of the detectable moietydetected and measured can be correlated to the amount of analyte presentin the test sample. For example, in a competitive immunoassay system, asubstance being measured, often referred to as a ligand, competes with asubstance of close structural similarity coupled to a detectable moiety,often referred to as a tracer, for a limited number of binding sites onantibodies specific to the portion or portions of the ligand and tracerwith structural similarity. These binding sites are usually shared withan immunogen employed to produce such antibodies.

Test Kits

A test kit according to the present invention comprises all of theessential reagents required to perform a desired specific fluorescencepolarization immunoassay according to the present invention for thequantification of thyroxine in a test sample. The test kit is presentedin a commercially packaged form as a combination of one or morecontainers holding the necessary reagents, as a composition or admixturewhere the compatibility of the reagents will allow.

Particularly preferred is a test kit for the fluorescent polarizationimmunoassay quantification of thyroxine in a test sample, comprisingfluorescent tracer compounds and antibodies as described above for thequantification of thyroxine. It is to be understood that the test kitcan, of course, include other materials as are known in the art andwhich may be desirable from a user standpoint, such as buffers,diluents, standards, and the like.

The present invention will now be illustrated, but is not intended to belimited by, the following examples. In Examples 1 and 2, the bold-facednumerals contained in parenthesis refer to the structural formulae asused in FIGS. 1 and 2, respectively:

EXAMPLE 1 SYNTHESIS OF THE L-THYROXINE IMMLINOGEN (7)

Abbreviations: EtOH=ethanol, NH₄ OH=ammonium hydroxide, HCl=hydrochloricacid, DMF=dimethylformamide, NaOH=sodium hydroxide, THF=tetrahydrofuran,CH₂ Cl₂ =methylene chloride, MEOH=methanol, HOAc=acetic acid.

L-Thyroxine sodium salt, pentahydrate (1)(10 g, 11 mmol) was nearlycompletely dissolved in 400 mL of EtOH/2 N NH₄ OH (1/1, v/v), filtered,and filtrate poured into 425 mL of 5% HCl. The resulting precipitate wasisolated by vacuum filtration and dried under high vacuum to afford awhite solid. This material was dissolved in 160 mL DMF, 100 mL (1.06mol) of acetic anhydride added, and reaction stirred for 1.5 hours, thendiluted with 850 mL H₂ O and allowed to stand at 4° C. for 16 hours. Theresulting precipitate was isolated by filtration, then dissolved in 350mL EtOH and 41 mL of 1N NaOH, stirred for 2.5 hours, 680 mL of 5% HCladded, and mixture allowed to stand at 4° C. for 16 hours. The resultingprecipitate was isolated by vacuum filtration and dried under highvacuum to yield 8.1 g (90%) of the desired N-acetyl L-thyroxine (5) as awhite solid; ¹ H NMR (200 MHz, CD₃ OD) d 7.8 (s, 2H), 7.1 (s, 2H),4.6-4.7 (m, 1H), 2.8-3.0 (m, 2H), 2.0 (s, 3H); mass spec (FAB)(M+H)+820.

N-Acetyl L-thyroxine (5) (1.0 g, 1.2 mmol) was dissolved in 50 mL THF,170 mg (1.5 mmol) of N-hydroxysuccinimide added, 300 mg (1.5 mmol) of1,3-dicyclohexylcarbodiimide added, and reaction stirred under N₂ for 3days. The reaction was then vacuum filtered to remove insoluble urea,affording 40 mL of filtrate. Half the filtrate volume (20 mL, 0.6 mmol)was combined with 80 mg (0.6 mmol) of 6-aminocaproic acid, reaction pHadjusted to 9 with triethylamine, and reaction allowed to stir under N₂for 2 days. Solvent was then removed in vacuo and crude product purifiedby elution on a silica gel Chromatotron® (Harrison Research, Palo Alto,Calif.), eluting with CH₂ Cl_(2/) MeOH/HOAc (90/10/0.2, v/v), to yield300 mg (54%) of the desired product (6) as a yellow oil; Mass spec (FAB)(M+H)+933.

The acid (6) (300 mg, 0.322 mmol) was dissolved in 25 mL THF, 45 mg(0.39 mmol) of N-hydroxysuccinimide added, 80 mg (0.39 mmol) of1,3-dicyclohexylcarbodiimide added, and the reaction mixture stirred for16 hours under N₂. The reaction was then vacuum filtered to removeinsoluble urea, affording 16 mL of filtrate. Then 4 mL (0.08 mmol) ofthe filtrate was added to a stirred solution of 250 mg (0.0037 mmol) ofbovine serum albumin dissolved in 10 mL of 0.05M sodium phosphate(pH=8.0) and 10 mL DMF. After stirring for 3 days the reaction wasdialyzed against 4 L of 0.05M sodium phosphate (pH=8.0) for 24 hours,then against 4 L of H₂ O for 24 hours, then lyophilized, to afford 297mg of the desired L-thyroxine immunogen (7).

EXAMPLE 2 SYNTHESIS OF THE L-THYROXINE TRACER (4)

Abbreviations: THF=tetrahydrofuran, EtOAc=ethyl acetate,DMSO=dimethylsulfoxide, CHCl₃ =chloroform, CH₂ Cl₂ =methylene chloride,MeOH=methanol, HOAc=acetic acid, Hex=hexane, DMF=dimethylformamide.

Sodium carbonate (7.85 g, 74.1 mmol) was dissolved in 480 mL H₂ O, 480mL THF added, 22.0 g (24.7 mmol) of L-thyroxine sodium salt,pentahydrate (1) added, 7.04 g (27.2 mmol) of 9-fluorenylmethylchloroformate added, and reaction stirred for 30 minutes. The reactionwas then diluted with 170 mL of 1M HCl and extracted with EtOAc (3×700mL). The EtOAc extracts were combined, dried over Na₂ SO₄, and solventremoved in vacuo to afford 26.3 g of the desired N-FMOC product as abeige solid; ¹ H NMR (300 MHz, DMSO-D6) d 9.29 (s,1H), 7.08-7.89 (m,12H), 4.19-4.29 (m, 4H), 3.06-3.16 (m, 1H), 2.82 (t, 1H); mass spec(FAB) (M-H+Na)+1021.

The N-FMOC protected L-Thyroxine (26.3 g, 23.2 mmol) was dissolved in150 mL THF, 3.28 mL (34.8 mmol) of acetic arthydride added, 283 mg (2.32mmol) of 4-dimethylaminopyridine added, and reaction stirred under N₂for 45 minutes, then poured into 400 mL H₂ O and extracted with CHCl₃(3×400 mL). The CHCl₃ extracts were combined, dried over Na₂ SO₄, andsolvent removed in vacuo. The residue was then purified by silica gelcolumn chromatography, eluting with CH₂ Cl_(2/) MeOH/HOAc (90/10/0.4,v/v), to yield 21.95 g (91%) of the desired O-acetyl thyroxine as abeige solid; ¹ H NMR (300 MHz, DMSO-D6) d 7.12-7.91 (m, 12H), 4.07-4.31(m, 4H), 3.07-3.19 (m, 1H), 2.82 (t, 1H), 2.29-2.40 (m, 3H); mass spec(FAB) (M+H)+1042.

The N-FMOC, O-acetyl L-thyroxine (21.70 g, 19.17 mmol) was dissolved in250 mL CH₂ Cl₂, cooled to 0° C., and 19.20 g (95.85 mmol) ofO-t-butyl-N,N'-diisopropylisourea in 50 mL CH₂ Cl₂ added in a dropwisefashion. The reaction was then stirred overnight, under N₂, at roomtemperature, then vacuum filtered to remove insoluble impurities, andfiltrate solvent removed in vacuo. The resulting residue was stirred in300 mL of EtOAc/Hex (40/60, v/v) for 4 hours, vacuum filtered to removeinsoluble impurities, and filtrate solvent removed in vacuo. The residuewas then purified by silica gel column chromatography, eluting withEtOAc/Hex (40/60, v/v), to afford 9.64 g (46%) of the desired t-butylester (2) as a beige solid; ¹ H NMR (300 MHz, CDCl₃) d 7.18-7.82 (m,12H), 4.21-4.57 (m, 4H), 3.05 (s, 2H), 2.39 (s, 3H), 1.33-1.54 (m, 9H);mass spec (FAB) (M+H)+1098.

N-FMOC, O-Acetate L-thyroxine t-butyl ester (2) (9.59 g, 8.04 mmol) wasdissolved in 40 mL DMF, 1.12 mL (8.04 mmol) of triethylamine added, andreaction stirred overnight, under N₂. Then 1.78 mL (16.1 mmol) of ethylbromoaoetate was added, followed by another 1.12 mL (8.04 mmol) oftriethylamine, and reaction stirred an additional 2 hours under N₂, thenpoured into 200 mL H₂ O and extracted with EtOAc (3×200 mL). The EtOAcextracts were combined, dried over MgSO₄, and solvent removed in vacuo.The resulting oil was purified initially by silica gel columnchromatography, eluting with EtOAc/Hex (40/60, v/v), and then purified asecond time by preparative silica gel HPLC, eluting with EtOAc/Hex(20/80, v/v), to yield 3.77 g (49%) of the desired N-carboxymethyl ethylester derivative as a white solid; ¹ H NMR (300 MHz, CDCl₃) d 7.75 (s,2H), 7.19 (s, 2H), 4.20 (q, 2H), 3.39-3.49 (m, 3H), 2.80-2.98 (m, 2H),2.39 (s, 3H), 1.42 (s, 9H), 1.27 (t, 3H); mass spec (FAB) (M+H)+962.

The ethyl ester intermediate (3.73 g, 3.88 mmol) was dissolved in 85 mLMeOH, 12.4 mL (31 mmol) of 10% sodium hydroxide added, and reactionstirred for 40 minutes. The reaction was then poured into 250 mL H₂ O,pH adjusted to 4 with 1M HCl, and extracted with EtOAc (3×250 mL). TheEtOAc extracts were combined, dried over MgSO₄, and solvent removed invacuo to afford 3.29 g (95%) of the desired product (3) as a whitesolid; ¹ H NMR (300 MHz, DMSO-D6) d 7.81 (s, 2H), 7,07 (s,.2H), 3.52 (t,1H), 3.32 (s, 2H), 2.89-2.98 (m, 1H), 2.20-2.31 (m, 1H), 1.32 (s, 9H);mass spec (FAB) (M+H)+892.

The free acid (3) (1.78 g, 2.00 mmol) was dissolved in 20 mL DMF, 230 mg(2.00 mmol) of N-hydroxysuccinimide added, 413 mg (2.00 mmol) of1,3-dicyclohexyicarbodiimide added, and reaction stirred for 16 hours,under N₂. The reaction was then vacuum filtered, filtrate combined with884 mg (2.00 mmol) of 5-aminomethylfluorescein hydrobromide and 1.8 mL(13 mmol) of triethylamine, and the reaction stirred for 16 hours, underN₂, in the dark, then solvent removed in vacuo. The residue was purifiedby preparative reverse phase C₁₈ HPLC, eluting with H₂ O/MeOH/HOAc(25/75/0.4, v/v), to afford 1.51 g (61%) of the desired t-butyl esterprotected tracer as an orange solid; ¹ H NMR (300 MHz, DMSO-D6) d 10.13(s, 2H), 9.29 (s, 1H), 8.43 (t, 1H), 7.85 (s, 1H), 7.83 (s, 2H), 7.68(d, 1H), 7.12-7.28 (m, 2H), 7.07 (s,. 2H), 6.68 (s, 2H), 6.54 (s, 4H),4.36-4.61 (m, 2H), 3.26-3.50 (m, 3H), 2.94-3.04 (m, 1H), 2.71-2.82 (m,1H), 1.33 (s, 9H); mass spec (FAB) (M)+ 1234.

The t-butyl ester tracer (1.464 g, 1.19 mmol) was dissolved in 30 mL ofCH₂ Cl_(2/) trifluoroacetic acid (1/1, v/v), stirred for 5 hours, andsolvent removed in vacuo. The crude product was purified by preparativereverse phase C₁₈ HPLC, eluting with H₂ O/MeOH/HOAc (25/75/0.4, v/v) toyield 1.01 g (72%) of the desired L-thyroxine tracer (4) as an orangesolid; ¹ H NMR (300 MHz, DMSO-D6) d 10.0-10.3 (broad s, 2H), 8.31 (t,1H), 7.86 (s, 2H), 7.83 (s, 1H), 7.64 (d, 1H), 7.15-7.30 (m, 2H), 7.08(s, 2H), 6.68 (s, 2H), 6.55 (s, 4H), 4.31-4.59 (m, 2H), 3.28-3.55 (m,3H), 2.82-2.99 (m, 2H); mass spec (FAB) (M+H)+1179.

EXAMPLE 3 ANTIBODY PRODUCTION Immunization Strategy

A 2 ml slurry mixture containing 1.0 mg of the lyophilized immunogen, asdescribed in Example 1, in physiological buffered saline (catalog no.#NDC 007-7983-02, Abbott Laboratories, Abbott Park, Ill., was added toMPL+TDM adjuvant solution as contained in a vial provided by themanufacturer (catalog no. #R-700, RIBI Immunochem Research, Inc.,Hamilton Mont.) and vigorously vortexed for 3 minutes. Fifteen mice ofstrain BCF1 (Jackson Laboratories, Bar Harbor, Me.) each received a 0.1ml injection dispersed equally between subcutaneous and interperitonealsites. This immunization strategy, repeated every 2 weeks for a total of4 boosts was followed by collection of serum samples 2 weeks later. Thebleed was allowed to incubate for 2 hours at room temperature before thesera was withdrawn and stored at -20° C. or colder.

Sera Evaluation

The serum Samples were tested on the TDx® instrument using thecommercially available T₄ reagent pack (both from Abbott Laboratories,Abbott Park, Ill.) to determine the presence of antibody which couldbind the commercially available TDx® T₄ tracer from the TDx® T₄ reagentpack (Catalog Code No. 97608, Abbott Laboratories) in a fluorescencepolarization assay. The screening assay was essentially the same asdescribed in the commercially available TDx® T₄ assay user manual exceptthat the T₄ antibodies in the commercially available T₄ reagent packwere replaced with the commercially available TDx® diluent (AbbottLaboratories.). The serum samples to be tested were added to the samplewell of the cartridge of the TDx® carousel. The serum samples weretitrated in log₂ dilutions in the sample wells of the TDx® carousel.Nine of the mice produced antibodies which bind the commerciallyavailable TDx® T4 tracer. One of the animals, designated animal #12, wasselected because its Net P (i.e. Net Polarization) signals were greaterthan the titrated normal mouse serum control from non-immunized mice(Catalog No. #5011-1380, lyophilized normal mouse serum, Cappel, Dunham,N.C.) by 60-80 mP. ("mP" denotes "millipolarization").

Fusion

Following a 5 month rest period, animal #12 was administered a 25 μg/mlinterveneous pre-fusion boost 3 days prior to the fusion. On the day ofthe fusion, the animal was sacrificed and the splenocytes were washedone time in Iscove's Modified Dulbecco Medium (IMDM) (GIBCO, GrandIsland, N.Y.) and centrifuged 1000 RPM for 10 minutes. The pelletedsplenocytes were combined with SP2/0 myeloma cells (from the laboratoryof Dr. Milstein, Cambridge, United Kingdom) at a 1:3 ratio, washed inIMDM, and centrifuged. The supernatant was removed and 1 ml of 50% PEG(i.e. polyethylene glycol) (American Type Culture Collection, Rockville,Md.) was added to the pellet for 1 minute as the pellet was gently beingdispersed by tapping and swirling. Thirty mls of IMDM was added to themixture and centrifuged as previously described. Supernate was decanted,the pellet was resuspended in IMDM with HAT (i.e. hypoxanthineaminopterin thymidine) (GIBCO), 10% FBS (i.e. fetal bovine serum)(Hyclone, Logan, Utah) and 1% STM v/v (RIBI Immunochem Research, Inc.).STM denotes Salmonella typhimurium mitogen. The STM solution was addedas a B-cell mitogen. The fusion cell suspension was plated into 96-welltissue culture plates.

Primary Fusion Screening

The primary screening of the fusion occurred on day 12 of confluentcultures. The Screen Machine (IDEXX, Portland, Me.) fluorescentconcentrated particle immunoassay (FCPIA) utilizes goat anti-mousemicroparticles to capture any mouse antibody secreted from the hybridsin supernate. The commercially available Abbott T₄ tracer (from the TDx®T₄ assay-reagent pack, Abbott Laboratories) was added to identify theantibodies which were T₄ reactive (i.e. bound T₄). Relative fluorescentintensity of hybrid #1-189 was 3 times that of the negative control(Catalog No. #5011-1380, lyophilized normal mouse serum, Cappel, Dunham,N.C.) and was selected as a candidate for further evaluation andcloning.

Hybrid Cloning

Hybrid #1-189 was cloned directly from the 96 well fusion plate bylimiting dilutions starting at 1-100, 10-fold to 10⁶. The cloning mediaused was IMDM with 10% v/v FBS and 1% v/v HT (i.e. hypoxanthinethymidine) Supplement (GIBCO). 100 μl cell suspension was added to eachof the 96 wells in the tissue culture plate. On day 7 the plates werefed with 200 μl/well of cloning media.

Clone Selection

Clone T₄ 1 -189-252 was selected for further evaluation based on themodified TDx® T₄ screen of the above "Sera evaluation" section. Thepolyclonal antisera in the T₄ reagent pack was replaced withcommercially available TDx® diluent (Abbott Laboratories.). The clonesupernate was added to the sample well of the cartridge of the TDx®carousel. In the pre-dilution well of the TDx® cartridge, duplicates of0 and 24 μl/dl free T₄ were placed. The TDx® T₄ Plus assay (previouslycommercially available from Abbott Laboratories for the TDx® instrument,this assay measured the total circulating T₄ in a serum or plasmasample) was run (as described in the previously commercially availableTDx® T₄ Plus assay user manual), and the monoclonal antibody sampleswhich showed a decrease in polarization were selected for furtherevaluation. The decrease in polarization was due to the T₄ in the samplewhich competitively displaced the T₄ -FITC from the monoclonalantibdody. For this experiment, the current commercially available TDx®T₄ assay (Abbott Laboratories, this assay also measures the totalcirculating T₄ in a serum or plasma sample) conducted according to itsassay user manual, could be used in place of the TDx® T₄ Plus assay. Thepolyclonal antibodies and tracer used in the TDx® T₄ Plus assay are thesame as those used in the TDx® T₄ assay.

Isotype

The isotype of the monoclonal antibody secreted from the cell lineidentified as T₄ 1 -189-252 was determined on an EIA clonotyping kit(Southern Biotech, Birmingham, Ala.). The assay was performed accordingto the vendor recommendations and the results indicated an isotype ofIgG2a, kappa.

Isoelectric Focusing

The isoelectric point (pl) of the monoclonal antibody secreted from thecell line identified as T₄ 1 -189-252 is determined on an isoelectricfocusing apparatus (Bio Rad, Richmond, Calif.). The gel was cast and runaccording to vendor recommendations. The results indicated pl=7.4±0.2.

Cell Line Deposit

The hybridoma cell line T₄ 1 -189-252 has been deposited with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. in accordance with the Budapest Treaty. Thedeposit date is Sep. 16, 1992 and the ATCC number assigned to the cellline is HB 11125. The monoclonal antibody produced by this hybridoma ishereby referred to as monoclonal antibody 1-189-252.

EXAMPLE 4 FLUORESCENCE POLARIZATION IMMUNOASSAY FOR THYROXINE

Endogenous antibodies present in the serum samples of certainindividuals cause falsely low total T₄ readings in the commerciallyavailable TDx®, TDxFLx™; and IMx® assays. These same samples, whenevaluated by the RIA method, yield values consistent with clinicaldiagnosis. Evaluation of these serum samples demonstrated that a bindingfactor, an immunoglobulin, is present that has a high affinity for thecommercially available Abbott T₄ tracer. This results in an increase inretention of polarization generating a high millipolarization units (mP)value, hence a low T₄ reading.

In the present invention, the tracer of Example 2 and monoclonalantibody 1-189-252 were optimized to perform as well as the commerciallyavailable Abbott T₄ FPIA, and it was discovered that this new assay hasthe additional advantage of avoiding the above discrepant readings. Thenew assay uses the same standard protocol and diluents as used in thecommercially available assays. The results of the assay run are reportedin millipolarization units (mP). The mP units are automaticallyinterpolated from a stored standard curve and expressed asconcentrations (microgram/dL) of thyroxine in the assayed samples. Thisprocedure is the same for both the commercially available Abbottreagents and the new tracer and monoclonal antibody.

For example, in the case of the TDx® new T₄ assay, the samples were runaccording to the standard protocol on the TDx® analyzer. The efficacy ofthe TDx® new T₄ assay was evaluated by comparing it with thecommercially available TDx® T₄ assay using 373 patient serum samples.Good agreement between the two assays for detecting T₄ was found. Thenew assay could detect between 0 to 24 μg/dL of thyroxine in a sample.Samples containing thyroxine concentrations greater than 24 μg/dL shouldbe diluted first, for example, in accordance with the vendor'sinstruction for the commercially available TDx® T₄ assay.

Further, for the TDx®, TDxFLx™, and IMx® new T₄ assays, the minimumpolarization span for the standard calibration curves is preferably atleast 100 mP, more preferably greater than 125 mP or 130 mP. Thecommercially available assays have similar spans. The upper limit forthe span is preferably less than 300 mP. To achieve the desired span,the tracer in the assays must bind to the antibody used and musteffectively compete with endogenous T₄ present in a sample.

Additionally, by measuring the ratio of the concentrations of T₄ totriiodothyronine (T₃) which correspond to the midpoint of thepolarization span for the T₄ standard curve, it was observed that thecross-reactivity of monoclonal antibody 1-189-252 with T₃ in the TDx®new T₄ assay was about 8%. Such low level of cross-reactivity isimportant because T₃ is another thyroid hormone in the human serum whichresembles T₄. Therefore, for an accurate assay of T₄, it is importantthat the antibody against T₄ does not substantially cross-react with T₃.Thus, such cross-reactivity is preferably about 15% or less, and morepreferably about 10% or less.

To compare the commercially available T₄ tracer and antibody to that ofthe present invention, a number of discrepant samples were run using thestandard protocol of the commercially available Abbott TDx® T₄ assay,using: 1) the commercially available TDx® T₄ tracer and antibody; 2) thecommercially available TDx® T₄ tracer, but monoclonal antibody 1-189-252in place of the commercially available antibodies; 3) monoclonalantibody 1-189-252 and the tracer of Example 2, in place of thecommercially available TDx® T₄ antibody and tracer. These samples werealso run on Abbott Tetrabead-125® radioimmunoassay (T₄ RIA, AbbottLaboratories) according to the vendor's recommended protocol.Information regarding the TDx® assay method can be found in the TDx®System Operation Manuals. The TDx® System Operation Manuals contain: 1)theory of operation: fluorescerise polarization immunoassay; 2)operational precautions and limitations; 3) daily start-up procedure; 4)monthly and periodic procedures necessary for quality control to bemaintained. The discrepant samples were obtained from patients whosesamples gave a reading of zero or abnormally low level of thyroxineconccentration in the commercially available TDx® T₄ assay but a readingindicating the presence of or a higher level of thyroxine concentraionin a T₄ RIA assay.

The results of the above assays are as follows:

                  TABLE 1                                                         ______________________________________                                        T.sub.4 (microgram/dL)                                                                                      3. TDx ®                                             1. TDx ®                                                                             2. TDx ®                                                                            T.sub.4 Assay                                            T.sub.4 Assay                                                                            T.sub.4 Assay                                                                           MAB                                                      Commercially                                                                             MAB       1-189-252 &                                              Available  1-189-252 &                                                                             Tracer of                                       DISCREPANT                                                                             Reagents   *T.sub.4 Tracer                                                                         Example 2                                                                             4. T.sub.4 RIA                          ______________________________________                                        1        1.97       0.0       9.80    10.26                                   2        0.0        0.0       6.78    7.02                                    3        2.21       0.0       6.17    6.41                                    4        1.04       --        7.97    9.39                                    5        0.0        0.0       7.03    8.23                                    6        0.0        0.0       5.34    4.63                                    7        0.0        0.0       12.75   15.95                                   8        0.0        0.0       8.82    10.16                                   9        2.65       2.24      7.03    8.18                                    10       0.0        0.0       15.85   15.43                                   11       3.31       0.89      7.78    8.71                                    12       2.37       0.0       8.57    10.05                                   ______________________________________                                         In this table, "*T.sub.4 tracer" denotes the commercially available           T.sub.4 tracer.                                                          

The above results demonstrate the effectiveness of the new tracer(column 3) yielding T₄ values within the expected range as compared tothe T₄ RIA result.

The tracers and antibodies of the present invention can also be used inthe IMx® and TDxFLx™ T₄ assays.

To determine whether the tracer was responsible: for the falsely low T₄reading in the discrepant samples, the following assays were conducted.The commercially available TDx® reagent pack contains three bottles ofreagents, designated "S", "T", and "P" pots. The S pot contains the T₄antibodies. The T pot contains the commercially available T₄ tracer T₄-FITC. The P pot contains the T₄ pretreatment solution which removes theprotein from any protein bound thyroxine in order to release thethyroxine for assay. In the following, the commercially available assaywas conducted with the exception that the S pot which normally containedthe T₄ antibodies was replaced with a buffer solution. First, adiscrepant serum sample (from one of the samples in Table 1) and anormal serum sample were tested and their results are shown in Table 2.As shown in Table 2, the assay gave a high mP value for the discrepantsample. In contrast, the normal sample generated a significantly lowermP value.

                  TABLE 2                                                         ______________________________________                                        mP VALUES IN THE PRESENCE OF COMMERCIALLY                                     AVAILABLE T.sub.4 TRACER AND                                                  ABSCENCE OF T.sub.-4 ANTIBODIES                                               SAMPLE         mP Value                                                       ______________________________________                                        Discrepant     206.13                                                         Normal Serum   195.92                                                         ______________________________________                                    

Then, the above described assay (wherein the S pot contained a bufferinstead of antibodies) was conducted on the same discrepant samples asused in Table 1 and the results were shown in Table 3 below. One run wasconducted with the commercially available tracer and one run wasconducted with the tracer of Example 2. A reduction in mP was observedwith the tracer of Example 2, demonstrating that the endogeneousimmunoglobulins in the discrepant samples do not bind to the tracer ofExample 2.

                  TABLE 3                                                         ______________________________________                                        Binding of Endogenous lmmunoglobulins by                                      Commercially Available Tracer and                                             Tracer of Example 2                                                           mP Value                                                                                    Commercially                                                                  Available  Example 2                                            Discrepant    Tracer     Tracer                                               ______________________________________                                        1             234.55     95.34                                                2             225.79     99.88                                                3             199.05     96.78                                                4             232.76     96.16                                                5             213.26     96.01                                                6             176.04     104.44                                               7             276.63     108.89                                               8             397.74     103.30                                               9             195.64     100.21                                               10            282.71     84.15                                                11            195.25     97.55                                                12            230.48     115.77                                               ______________________________________                                    

As described above, an evaluation of these discrepant serum samplesdemonstrated that the binding factor present was an immunoglobulinhaving an affinity for the commercially available T₄ tracer. Thedetermination was accomplished by the following methods: HPLC,immunoblot analysis and protein G sepharose separation.

In the HPLC study, proteins found in discrepant patients were separatedby HPLC using an anion exchange column. Fractions that had the abilityto bind the T₄ tracer were isolated. These fractions corresponded to theregion of the chromatographic profile where IgG would elute.

In addition, Immunoblot analysis of the selected fractions indicated aband corresponding to human IgG which was detected with goat α-humanIgG.

These selected fractions were then incubated with Protein-G sepharose.Protein-G sepharose selectively binds IgG. Following removal of theProtein-G sepharose by centrifugation, an analysis of the supernatantsrevealed that the T₄ binding component was no longer present.

It would be clear to one skilled in the art that the assays utilizingthe tracers and antibodies of the present invention can also beconducted on other FPIA instruments besides TDx®, TDxFLx™, and IMx®. Theparameters, such as the span and concentration of thyroxine detectable,will be optimized according to the characteristics, such as thesensitvity, of each instrument used.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if each ofthem had been individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and examples for purposes of clarity andunderstanding, various modifications and changes which are within theskill of those skilled in the art are considered to fall within thescope of the appended claims. Future technological advancements whichallow for obvious changes in the basic invention herein are also withinthe claims.

We claim:
 1. A method of making an immunogen of the formula: ##STR10##wherein BSA denotes bovine serum albumin, the degree of substitution ofBSA by the thyroxine derivative is between 1% to 100%, the thyroxine isan L-isomer, and antibodies directed against the immunogen immunoreactwith an L-thyroxine conjugate of the formula: ##STR11## where Q is adetectable moiety and W is a linking moiety, the method comprising thesteps of:(a) N-acetylating L-thyroxine to form α-N-acetyl L-thyroxine;(b) conjugating the αN-acetyl L-thyroxine to 6-aminocaproic acid to forma L-thyroxine conjugate; and (c) conjugating the L-thyroxine conjugateof step (b) to bovine serum albumin.